Pseudomonas aeruginosa is an important opportunistic pathogen of humans that is notorious for being the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients. In the chronically infected CF lung the organism persists as a biofilm-a surface attached community of bacteria encased in a polymeric matrix. This biofilm mode of growth augments the resistance of P. aeruginosa to antibiotics and facilitates evasion of the host immune response. Prominent amongst those genes that play an important role in biofilm formation in P. aeruginosa are the cupA genes, which encode components of a fimbrial structure that facilitates surface- attachment and host colonization. We have identified three genes (cgrABC), whose products are required for phase-variable (i.e. reversible ON/OFF) expression of the cupA fimbrial gene cluster. None of the products of the cgr genes resembles any classical positive regulator of gene expression; cgrA is predicted to encode a member of the adenine nucleotide -hydrolase superfamily, whereas cgrB encodes a putative acetylase, and cgrC encodes a protein with homology to the ParB family of DNA-binding proteins. In Aim 1 we propose to determine how the Cgr proteins exert their control. We will explicitly test the hypotheses that CgrB functions by acetylating CgrA and that phase-variable expression of the cupA genes is mediated by changes in the acetylation state of CgrA. Because many other pathogenic bacteria contain cgr orthologs, we expect that the relevance of our studies will extend beyond P. aeruginosa. Recently, we identified PrrA, a novel small regulatory RNA (sRNA), as an additional positive regulator of the cupA genes. Preliminary evidence suggests that PrrA exerts its effects on cupA expression by antagonizing the silencing effects of a member of the H-NS family of histone-like nucleoid structuring proteins. In Aim 2 we propose to determine how PrrA functions as an anti-silencer. We anticipate that the proposed studies will enable us to (i) define roles for protein acetylation and an sRNA in the control of phase-variable virulence gene expression in P. aeruginosa and (ii) determine precisely how the local and global regulatory networks that influence cupA gene expression become effectively integrated.